Method for treating collagen fibers



United States Patent 3,231,463 METHGD FOR TREATKNG COLLAGEN FIBERS Roger de la Burde, Chicago, and Arthur Veis, Skokie, Ill, assiguors, by mesne assignments, to Armour and Company, Chicago, IEL, a corporation of Delaware No Drawing. Filed Oct. 31, 1960, Ser. No. 65,885 The portion of the term of the patent subsequent to Sept. 25, 1979, has been diselaimed 7 Claims. (Cl. 162-2) This invention relates to the disaggregation of collagen. Particularly it relates to chemically modified fibers of collagen and to a method for preparing them.

Collagen is the primary intercellular protein constituent of the white connective tissue in animal tissues. Skin, tendons, reticular tissue, bones and various other animal structures contain interwoven networks of collagen fibers which are highly organized in the native structures. Hydrolysis and other drastic chemical treatments normally melt or destroy the organized structure without releasing fibers in an intact form. Each fiber is ordinarily locked within the woven structure and tenaciously adheres to contiguous fibers.

Chemical modification, or tanning, of collagen to produce leather is traditionally performed on intact animal skins. During fabrication of leather goods, scrap leather resulting from the fabrication, plus defective skin areas which must be discarded, represents economic loss to the fabricator. It is therefore highly desirable to have available homogeneous collagenous raw material which may be reassembled into desirable structures without formation of waste material and which utilizes leather scrap. Because cost of fabrication is further increased by variations in thickness and shape of leather pieces, it is desirable to have available a method for reassembling collagen into skin-like structures of uniform thickness and shape.

Previously, attempts have been made to prepare collagen structures, such as sheets, from reconstitutable collagen. Heretofore the most promising method of preparing reconstituted collagen involves dissolving the procollagen fractions of collagen and re-precipitating these dissolved fractions in the presence of undissolved fibers to give the resulting mass strength and cohesiveness. Difiiculties have been encountered in attempting to obtain strong and intact undissolved fibers. Previously, native collagen has been mechanically beaten to break fibers from the organized collagen structure. This mechanical beating results in cutting of the fibers, the shorter fibers in turn result in formation of lower strength reaggregated materials.

We have discovered that by selective chemical action the forces which hold collagen fibers together in laminated form may be destroyed without substantially affecting the physical structure of the individual fibers, thus retaining the original long fiber formation found in the native collagen aggregates.

Our invention provides a method for treating collagen so that the interwoven fibers may be slipped from the aggregated or native structure in an essentially intact state. The intact but disaggregated fibers which have been chemically modified to render them highly reactive may then be rewoven into desired structures. This invention also provides a method for reweaving or reaggregating collagen.

Therefore an important object of this invention is to provide a method for preparing disaggregated intact collagen fibers which are susceptible to reaggregation. A second object is to provide dispersed long collagen fibers which have been selectively modified by the introduction of highly reactive chemical groups. Another object is to provide a method for utilizing previously unsuitable starting materials as leather stock. Still a further object is to 3,231,463 Patented Jan. 25, 1966 provide a method for converting collagen into raw stock suitable for the manufacture of all types of leather articles. One advantage of this invention is to make available for leather manufacture heretofore unsuitable collagen sources. Another advantage is the profitable utilization of leather scrap from fabricating operations. A further advantage is the elimination of low quality leather stock. Other objects and advantages will become apparent in the following description of our invention.

Basically this invention is concerned with the treatment of collagen by hydrazine for a suitable period of time and at suitable concentrations to cause separation of collagen fibers from one another without rupturing the structure of the fibers themselves. Hydrazine conditioning causes the intact fibers to slip apart from one another, making mechanical disaggregation of the collagen fibers feasible. Following mechanical disaggregation, the action of the hydrazine is stopped and the fibers may be further treated to prepare them for reaggregation back to a highly organized collagen structure.

Various sources of collagen are suitable starting materials for the practice of our invention. Fresh and dehydrated hides and skins, enzymatically treated hides, detanned leather, ichthyol, tendons, and ossein are examples of materials which may be used. Preferably limed or unlimed hides which have been partially purified as by defatting, salt removal and extraction with acid and base solutions, are used as raw materials.

We have found that hydrazine has the surprising property of reacting with collagen in a manner which renders the aggregated collagen fiber mass susceptible to easy mechanical disaggregation. Fibers in the mass retain their individual physical structure while losing their ability to adhere to one another in the manner existing in native collagen aggregates. The action of hydrazine is contrasted with the action of other types of reagents such as bases which initially cause swelling of the gross structure followed by rapid solubilization. Swelling causes the fibers to become more tightly bound to one another. Upon solubilization, clumps of the fibrous aggregate disorganize, resulting in the fibers themselves being ruptured. Hydrazine apparently reacts with certain functional groups of collagen, causing formation of a smooth, nonadheren-t surface on each fiber, with resultant slipping of the individual fibers within the aggregate.

Typically in the practice of the present invention, purified collagenous material is contacted by hydrazine solution. Hydrazine conditioning continues until forces creating adhesion between the fibers is broken. However, the conditioning is stopped before the individual fibers are ruptured or dissolved.

After suitable conditioning, the individual fibers of the collagen may be slipped from the organized structure by gentle mechanical agitation to form a slurry of relatively intact collagen fibers. Immediately before or after the mechanical agitation, the conditioning reagent should be removed from the conditioned collagen. We prefer to wash the fibrous slurry after mechanical agitation with sufiicient quantities of Water to remove substantial portions of the hydrazine. The separated fibers are then collee-ted after washing and may be further treated and reaggregated to reform a highly organized network structure.

We find that a solution of hydrazine having a concentration of about to 95 percent hydrazine by weight in water may be satisfactorily employed as the conditioning reagent. Preferably a concentration of about to 63 percent hydrazine is allowed to contact the collagen for a period of about 24 hours at room temperature. Length of conditioning is dependent upon the concentration of the hydrazine, the temperature employed and the character of the aggregated collagen. Temperatures below about C. should be maintained throughout the conditioning period. We prefer to maintain conditioning temperatures of to 60 C. is satisfactory. Conditioning time should at between 20? to 40 C. although a temperature range be shortened at higher temperatures to avoid fiber solubilization.

' Other suitable methods in lieu of water washing to remove or neutralize the hydrazine may be employed. One such method which may be used involves neutralization of the hydrazine by acid. Ordinarily we prefer to recover the hydrazine, recycle it and reuse it in the conditioning of subsequent batches of collagen.

During conditioning it is important that the collagenous material not be so rigorously conditioned as to cause excessive solubilization. Solubilization of the collagen, which may result from excessively long conditioning times and use of temperatures above about 60 C., should be avoided because it reduces the yield of intact fibers. In addition, solubilization results in formation of gels which act as undesirable impurities when mixed with the fibers. The gels formed during solubilization tend to glue the fibers together, often making their removal advisable before further treatment of the fibers. Proper conditioning causes satisfactory slipping of the fibers long before apprcciable solubilization occurs. The conditioning is allowed to progress to a point at which the forces linking the individual fibers into three-dimensional cross-linked networks are inactivated, thereby breaking the bonding between fibers. The selective chemical action is stopped before appreciable quantities of fibers are solubilized or physically disrupted.

Following conditioning, the aggregated collagen is disaggregated and slurried by gentle mechanical agitation.

Optimal slurrying manipulation may be described as crushing agitation rather than shearing manipulation. It is important in reconstructing high strength collagen structures that long fibers be available for reweaving or reaggregating. Grinding and shearing of the conditioned fiber reduces fiber length which often results in lower strength reaggregates. We have found that a Hollander type paper pulp beater produces excellent results in slurrying the conditioned collagen without disrupting the individual fibers. This type of beating machine is fitted with a roll and bedplate both having horizontal bars. Examples of satisfactory heaters include Sharples, Horne, Rabus, Umpherston, and Bertrams heaters. Other suitable types of mechanical equipment which do not cause excessive shearing may be used.

After suitable mechanical disaggregation the action of the hydrazine may be stopped by removal of it from the slurry, for example by water washing or some other method, and the fibers collected. Reaggregation of the collected fibers may be accomplished immediately after collection, or the fibers may be stored for long periods of time prior to further processing. If the fibers are to be retained for long periods of time before processing, growth of microorganisms should be inhibited by common wellknown methods such as drying, chemical stabilization and refrigeration.

Several desirable but unessential steps may be included during the processing of the collagen to increase the efiiciency of the operation. We have found that preconditioning or post-conditioning of the collagen material with alkaline solutions reduces the conditioning time and concentration of the hydrazine required. We prefer to postcondition the hydrazine treated collagen by exposing it to a dilute solution of a base such as 2 percent sodium hydroxide solution for a few hours. For example, after conditioning the starting material with hydrazine for several hours, sodium hydroxide may then be added to the reaction mixture and the mixture held for an additional few hours. The auxiliary alkali conditioning allows the use of more dilute hydrazine solutions. It must be remembered that prolonged contact with the strong bases such as sodium hydroxide may cause hydrolysis of the protein material and consequently careful control of the alkaline auxiliary treatment should be observed.

We also prefer to dehydrate the fibers after conditioning reagent removal. Several methods of dehydration may be used, both mechanical and chemical, but we prefer to use a solvent to partially dehydrate the fibrous slurry. Any of the well-known dehydrating solvents such as acetone, ethers and alcohols may be used. After dehydration, we also prefer to subject the collagen fibers to light pretannage to remove additional water. Contacting the partially dehydrated fibers with formaldehyde solution having a concentration of about 0.3 to 3 percent produces a satisfactory pretannage. Other well-known methods of tanning, such as vegetable and chrome tannage may be used.

To increase the economic efficiency of this process, we prefer to recover the hydrazine and reuse it in the conditioning of later batches of collagen. About of the hydrazine may be recovered by distilling a water solution separated from the fibers after disaggreation.

Collagen fibers resulting from the practice of our invention may be utilized in various ways, but we contemplate the most important use of these fibers to be their reaggregation into cross-linked and skin-like structures of organized collagen. Reaggregation of the separated fibers may be accomplished in several ways. Excellent results may be obtained by introducing cross-linking agents such as aliphatic mono and poly aldehydes, aromatic mono and poly aldehydes, chrome, salts, aluminum salts and various natural and synthetic tannins.

The disassembled fibers of this invention are modified by reaction with hydrazine to form highly reactive intact fibers which may be aggregated to form native or highly organized structures. Some groups contained in the native fibers, such as amides and guanidine groups of arginine, are replaced or removed by hydrazine. The new fiber products thus formed have essentially the same principal properties as native collagen fibers, although they are chemically modified. This particular modification of the individual fibers increases their reactivity and potentiality for crosslinking, dyeing, tanning, fat liquoring, finishing, and so on. One effect of modification, for example, is the introduction of additional hydrazide groups into the collagen, consequently increasing its reactivity toward acid dyes.

Modifying the collagen fibers as hereinbefore described results in amide removal and substitution by hydrazide groups. Each amide group that is removed is replaced by a hydrazide group which is covalently bonded, the substitution occuring at the rate of about 8 to 20 moles of hydrazine per 10 gms. of native collagen. In modified collagen a total of about 15 to 45 moles of hydrazine react with 10 gms. collagen. Guanidyl groups of the arginine residues are removed as aminoguanidine in the range of about 1 to 20 moles per 10 gms. collagen. By the conversion, 1 to 20 moles of arginine are converted to ornithine.

The delaminated and separated fibers of this invention retain the typical physical properties of collagen although they are modified by the hydrazine. They exhibit the usual cross striations of collagen, having an axial periodicity of 600 to 650 A. when viewed by means of the electron miscroscope.

The following detailed examples will serve to illustrate our invention.

Example I Dehydrated intact cattle hides were covered with a 60 percent aqueous solution of hydrazine at the rate of 4 parts hydrazine solution to 1 part of collagen by weight at about 25 C. The mixture was allowed to stand without mixing or agitation for 30 hours. At the end of this conditioning the collagen became opaque and was removed from the hydrazine bath. This material was then water washed briefly, and the mass was gently macerated,

to form a collagen slurry. Dissolved material and hydrazine was removed by further water washing. The hydrazine was regenerated by distillation for further use.

Example II A collagen slurry was obtained as in Example I and was Water washed briefly to remove the major part of the hydrazine absorbed by the collagen pieces. The hydrazine was then removed and distilled for use. Slurry was then water washed continuously under rapid agitation. After about 30 minutes at 25 C. a deswelling of the fibrous structure was noted, accomplished by whitening of color in the collagen slurry. The slurry was then washed in .5 percent formaldehyde. This was followed by water washing for 3 minutes, after which the fibers were centrifuged 01f and stored in the wet state at refrigerator temperatures.

Example III Dehydrated bovine corium was immersed in a solution of 60 percent hydrazine and 40 percent water which was maintained under agitation at a temperature of 25 C. After 20 hours the collagenous mass was pressed through a sieve, converting the collagen mass into a heavy opaque paste. This pressing operation was repeated several times. Collagen pieces which were too intact to pass through the sieve were removed and treated again with fresh hydrazine solution. The hydrazine solution which had been drained from the reacted collagen was subjected to distillation in vacuo. The collagen paste was water washed with running tap water. After minutes of water washing, the slurry was washed in 3 percent formaldehyde solution for minutes. The slurry was then water washed again until the original opaque pinkish-red color turned into a white color. This mixture of collagen fibers was transferred to a filter press and pressed to remove the bulk of water. The fibers which remained were placed in forms and pressed into sheets of desired thickness. The sheets were treated with acetone without removing them from the frame. Pressure then was exerted upon the frame for about 5 minutes, after which the collagen sheets were easily removed. The sheets were ready for tanning or lyophilization.

Example IV One pound of lirned dehaired cattle hide was immersed in a 5 liter bath of 23 percent hydrazine at a temperature of 25 C. The collagen was conditioned in the hydrazine for 24 hours with gentle agitation. Sufficient sodium hydroxide was then added to attain a concentration of 2 percent hydroxide in the conditioning bath. After 8 hours, the collagen was removed from the bath and gently macerated. The resulting fibers were thoroughly water washed.

Example V Using the procedure of Example I bull corium was disassembled in an 80 percent solution of hydrazine at a temperature of 7 C. for 10 hours. A satisfactory col lagen slurry resulted.

Example VI As in Example I bovine corium was conditioned in 40 percent aqueous hydrazine solution at a temperature of 50 C. for 15 hours to prepare a slurry of disassembled collagen fibers.

While in the foregoing specification various aspects of this invention have been described in considerable detail for the purpose of illustration, it will be apparent to those skilled in the art that our invention is susceptible to other embodiments and that many of these details can be varied 6 widely without departing from the basic spirit and concept of the invention.

We claim:

1. A process comprising the steps of conditioning aggregated collagen fibers with hydrazine to thereby induce disaggregation of long fibers in said collagen, mechanically disaggregating said fibers, separting portions of hydrazine from said fibers and collecting said fibers.

2. A process for preparing disaggregated intact collagen fibers comprising the steps of contacting collagen with a water solution of hydrazine to thereby induce disaggregation of fibers in said collagen, mechanically disaggregating said fibers, separating substantial portions of said hydrazine from the fibers, and collecting the fibers.

3. The method for preparing native collagen structures comprising the steps of contacting aggregated collagen fibers with hydrazine to thereby induce disaggregation of said fibers, mechanically disaggregating said fibers, removing substantial quantities of hydrazine from said fibers, collecting said fibers and reaggregating said fibers to form a highly organized collagen aggregate.

4. The process for treating collagen comprising the steps of contacting aggregated collagen with hydrazine, maintaining contact for sufiicient time to break bonding forces between fibers and said collagen, stopping action of the hydrazine before appreciable solubilization of said fibers occurs and collecting said fibers.

5. The process for treating collagen comprising the steps of contacting native collagen with a hydrazine solution having a concentration from about 20 to percent hydrazine by weight, maintaining contact for a period of time sufiicient to induce disaggregation of fibers in said collagen, mechanically disaggregating the fibers in said collagen, removing substantial quantities of said hydrazine from said fibers and collecting said fibers.

6. A method for preparing separated and essentially intact fibers of collagen comprising the steps of preparing a water solution of hydrazine, increasing alkalinity of said solution by the addition of a basic material thereto, conditioning aggregated collagen by contact with said solution to thereby induce disaggregation of fibers, mechanically disaggregating said fibers, preventing further conditioning action by said solution, and collecting said fibers.

7. A method for preparing separated and esentially intact fibers of collagen comprising the steps of preparing a water solution of hydrazine, preparing a water solution of an alkaline material, alternately contacting aggregated collagen with the solutions to thereby induce disaggregation of said fibers in said collagen, mechanically disaggregating said fibers, stopping action of said solutions before appreciable solubilization of said fibers occurs, and collecting said fibers.

References Cited by the Examiner UNITED STATES PATENTS 2,105,035 1/ 1938 Freudenberg et a1. 19-2 2,115,648 4/1938 Runkel 192 2,266,907 12/1941 Riehl 2881 2,271,184 1/ 1942 Dreyfus 2881 2,382,632 8/1945 Hessel et al 8-127.6 3,055,790 9/1962 De La Burde 1622 OTHER REFERENCES OFlaherty, Chemistry and Technology of Leather, pub. by Reinhold, N.Y., N.Y. 1956, pp. 264, 283, 284.

DONALL H. SYLVESTER, Primary Examiner.

RUSSELL C. MADER, RICHARD N. NEVIUS,

MORRIS O. WOLK, Examiners. 

1. A PROCESS COMPRISING THE STEPS OF CONDITIONING AGGREGATED COLLAGEN FIBERS WITH HYDRAZINE TO THEREBY INDUCE DISAGGREGATION OF LONG FIBERS IN SAID COLLAGEN, MECHANICALLY DISAGGREGATING SAID FIBERS, SEPARATING PORTIONS OF HYDRAZINE FROM SAID FIBERS AND COLLECTING SAID FIBERS. 